Despite extensive studies over many years, the molecular and biochemical mechanisms of insulin action remain to be elucidated. The first step in this mechanism is the binding of insulin to a specific receptor on the plasma membrane of target cells. Analysis of the structure and function of the insulin receptor (IR) is hampered by the fact that all cells tested have detectable levels of endogenous IRs. We propose to take a novel approach by using homologousrecombination, following microinjection of mutated genomic DNA fragments, to inactivate the endogenous IR genes. Specifically, both single and double allele mutants will be produced in HepG2 hepatoma, and 3T3-F442A adipocyte cell lines. The single allele mutants will be used for studies involving the regulation of individual alleles, the double allele mutants, which will be receptor null cells, will be used for transfection studies with mutant IRs generated by in vivo site-directed mutagenesis. Furthermore, we will make functional mutations by in vivo mutagenesis, in one allele only to study, the interaction between IRs in a heterozygote cell as a model for NIDDM.